Association between P2Y1 and P2Y12 polymorphisms and acute myocardial infarction and ADP-induced platelet aggregation

Background The objective of this study was to investigate the relationship between P2Y1 and P2Y12 genotypes and the risk of acute myocardial infarction (AMI) in the Quanzhou population and to determine associations between P2Y1 and P2Y12 genotypes and ADP-induced platelet aggregation in this population. Methods All subjects were screened for P2Y1 (c.1622A > G) and P2Y12 (H1/H2, c.34C > T) polymorphisms by direct DNA sequencing. The maximal platelet aggregation rate (MAR) in AMI patients (n = 61) and healthy control subjects (n = 50) was measured by a PL-12 platelet function analyzer, and adenosine diphosphate (ADP) (5 μmol/L) was used as an agonist. Results The haploid H2 allele in the P2Y12 gene was more frequent in patients with AMI than in control subjects (OR 1.887, P = 0.005). The P2Y12 H2 haplotype was significantly associated with AMI in the codominant (P = 0.008), dominant (OR 2.103, P = 0.003), and overdominant models (OR 2.133, P = 0.003). After adjusting for potential confounders, H2 haplotype carriers had a 2.132-fold increased risk for AMI (OR 2.132, P = 0.012) compared with noncarriers. Moreover, we observed that the ADP-induced MAR in the carriers of the H2 haplotype from the control group was somewhat higher than that in noncarriers of this group (P = 0.020). However, we failed to demonstrate that the P2Y1 H1/H2 polymorphism affected ADP-induced MAR in AMI patients. Additionally, P2Y1 c.1622A > and P2Y12 c.34C > T polymorphisms were not associated with the risk of AMI or ADP-induced MAR in either group. Conclusions Therefore, our results suggest that the P2Y12 H2 haplotype was associated with a higher risk of AMI, while its effect on increased ADP-induced platelet aggregation remains to be investigated. Thus, the P2Y12 H2 haplotype may be a potential marker for AMI.


Introduction
Acute myocardial infarction (AMI) is a major cause of morbidity and mortality among cardiovascular diseases (CVDs) in China and is considered a specific type of coronary artery disease (CAD). CAD is a multifactorial disease attributed to the combination of conventional environmental risk factors and genetic factors. Polymorphisms in various genes have been suggested to be involved in the pathogenesis of CAD and influence its susceptibility [1]. Among them, the genetic polymorphisms of P2Y1 and P2Y12, which encode the P2Y1 and P2Y12 receptors, respectively, were found to be associated with thromboembolic events and altered responsiveness to platelet inhibitors [1][2][3][4], and they enhance platelet reactivity [5][6][7][8]. P2Y1 and P2Y12 are membrane receptors that participate in the process of platelet activation and aggregation via the combination of adenosine diphosphate (ADP), which is considered to be critical for the pathophysiology of atherosclerosis and its complications (e.g., stroke and myocardial infarction).
The P2Y1 gene maps to chromosome 3q21-q25 and consists of a single exon encoding 373 amino acid residues [6,9]. Several studies confirmed that the c.1622A > G polymorphism of the P2Y1 gene was associated with enhanced platelet reactivity and the antiplatelet drug response [2,6,10], but these findings were not confirmed in subsequent studies [7,[11][12][13]. Moreover, no study has addressed the impact or association of the c.1622A > G polymorphism in the P2Y1 gene and the risk of AMI.
The P2Y12 receptor plays an important role in promoting thrombosis and hemostasis to cerebrovascular events [14]. Deficiency of the P2Y12 receptor impairs coagulation and results in a tendency toward bleeding [15]. The P2Y12 gene is located on chromosome 3q21-q25, and exon 2 encodes the human P2Y12 receptor consisting of 342 amino acid residues. Initially, Fontana et al. first found three introns (i-139C > T, i-744 T > C, i-ins801A) and two exons (c.52G > T and c.34C > T) in the P2Y12 gene [5]. Among these gene polymorphisms, four (i-139C > T, i-744 T > C, i-ins801A, c.52G > T) were shown to be in absolute linkage disequilibrium and were designated haploid H1 (not carrying all four polymorphisms) and haploid H2 (carrying all four polymorphisms) [5]. Several reports have determined that the P2Y12 H2 haplotype is associated with increased platelet aggregation in response to ADP [5,8] and is associated with a high risk of CAD, ischemic stroke (IS), cerebral infarction and peripheral artery disease [1,3,16,17]. However, these associations were not confirmed in subsequent studies [18,19]; thus, its role remains unclear.
Few studies have described the relationship between P2Y1 and P2Y12 polymorphisms and AMI. Moreover, the effect of P2Y1 and P2Y12 polymorphisms on the extent of ADP-induced platelet activation in the Quanzhou population in China remains unknown. Therefore, the objective of our study was to investigate the association between the polymorphisms of P2Y1 (c.1622A > G) and P2Y12 (c.34C > T and H1/H2 haplotype) and the risk of AMI and their effect on ADP-induced platelet aggregation.

Study population
Our study recruited patients with AMI (n = 189) from the cardiovascular medicine department of Quanzhou First Hospital in Fujian Province from September 2020 to June 2021. These individuals included 108 males and 81 females (age range from 30 to 83 years). Patients with AMI were diagnosed by medical records, physical examination, electrocardiogram and three-dimensional echocardiography. Moreover, we enrolled age-and sexmatched individuals (n = 194) without a history of cardiovascular and cerebrovascular diseases (e.g., stroke, MI, or CAD) and family history of cardiovascular and cerebrovascular disease as control subjects. The control subjects included 105 males and 89 females (age range from 34 to 81 years). These studies were performed in accordance with the ethical guidelines under protocols approved by the Ethics Committee of Quanzhou First Hospital (Approval Number: [2018]221). Informed consent was obtained from all individual participants included in the study.

Measurement of ADP-induced platelet aggregation
For the aggregation analysis, we collected venous whole blood into tubes containing 3.2% sodium citrate from 61 random AMI patients and 50 healthy volunteers. None of the healthy volunteers smoked, consumed alcohol, took any antiplatelet medicines or suffered from diabetes mellitus or hypertension. Platelet aggregation was measured by a PL-12 platelet function analyzer (SINNOWA Medical Science & Technology Co., Nanjing, China) based on a new platelet count drop method, as described previously [20]. In brief, after the citrated sodium blood sample was placed into the measuring position, the PL-12 counted platelet number prior to and after mixing with inducer ADP (final concentration: 5 μmol/L). PL-12 was counted several times until the lowest level was detected. The maximal platelet aggregation rate (MAR) ratio was calculated using the following formula: MAR = (baseline-lowest platelet count) *100%/baseline.

Statistical analysis
The results are presented as the mean ± SEM unless indicated otherwise. Quantitative data between two groups were assessed using Student's t test, and qualitative data were assessed with a χ2 test or Fisher's exact test. The deviation of the Hardy-Weinberg equilibrium for all genotype frequencies in the present study was assessed by χ2 test. The relationship between the variables and AMI was studied using multivariate logistic regression analysis. Statistical analyses were performed using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was considered statistically significant. Table 1 demonstrates the demographic and clinical characteristics of the AMI patients and control subjects. Our study found that the body mass index (BMI), blood glucose levels, and triglyceride (TG) levels in patients with AMI were significantly higher than those in the control group, while their high-density lipoprotein cholesterol (HDL-C) level was lower than that of the control group (P < 0.05). Moreover, other traditional risk factors, such as hypertension and diabetes mellitus, were more prevalent in AMI patients than in control subjects (P < 0.05). However, no significant differences (P > 0.05) were found between the two groups in terms of age, sex, smoking status, alcohol intake, low-density lipoprotein cholesterol (LDL-C) levels, or total cholesterol (TC) levels.

Associations between P2Y1 and P2Y12 polymorphisms and AMI risk
The genotypic distributions of th 3 variants were in accordance with Hardy-Weinberg equilibrium (P > 0.05) ( Table 2). The distribution of the P2Y1c.1622A > G, P2Y12 c.34C > T and P2Y12 i-744 T > C genotypes and alleles in the control and AMI groups are listed in Table 3.
No significant association was observed between the P2Y1 c.1622A > G polymorphism and the incidence of AMI in the codominant model (P = 0.536), the dominant model (P = 0.895), the recessive model (P = 0.318), and the overdominant model (P = 0.469). Moreover, the frequency of the G allele in AMI patients was 26.7%, which was similar to that in control subjects (G allele occurred in 27.8%, P = 0.792) ( Table 3).
For the P2Y12 gene, the H1/H2 haplotype and c.34C > T polymorphism were selected for analysis in our study. The genotypic frequencies of the P2Y12 H1/ H2 haplotype were significantly associated with AMI  The genotypic distribution of c.34C > T, the other P2Y12 polymorphism, was not found to be associated with the occurrence of AMI in the codominant model (P = 0.383), the dominant model (P = 0.251), the recessive model (P = 0.670), and the overdominant model (P = 0.171). Moreover, the frequencies of the C and T alleles in AMI patients were 73.2% and 26.7%, respectively, which were similar to those in control subjects (C allele occurred in 79.4%, T allele occurred in 20.6%, P = 0.442).

Logistic regression analysis of risk factors for AMI
In the multiple regression analysis, we included the prediction of BMI, hypertension, diabetes, TG level, HDL-C level and the P2Y12 H1/H2 genotype. Logistic regression analysis showed that BMI, hypertension, diabetes and haploid H2 allele carriers were important independent regulatory factors for AMI (P < 0.05) ( Table 4). Individuals who were haploid H2 allele carriers had an ~ 2.054fold increase in the development of AMI compared with noncarriers after adjustment for other covariates (OR 2.132, 95% CI 1.180-3.854, P = 0.012).

Discussion
The possible role of genetic variations in the P2Y12 and P2Y1 genes in AMI susceptibility has not yet been thoroughly investigated. Moreover, their effect on ADPinduced platelet aggression remains elusive. In this study, we found that the P2Y12 H2 haplotype was associated with a higher risk of AMI. Additionally, the P2Y12 H2 haplotype displayed markedly increased ADP-induced MAR in healthy adults, but their association was not confirmed in AMI patients. In our study, the frequency of the 1662G allele was 27.8%, which is consistent (P > 0.05) with the findings in Korea (31%) and in the Hunan Province of China (30.7%) ( Table 6) [7,20]. In the analysis of Yi et al. [21], the P2Y1 c.1622A > G polymorphism was not associated with IS susceptibility. Supported by this finding, our findings indicated that the P2Y1 c.1622A > G polymorphism did not contribute to the risk of AMI. However, we failed to demonstrate that the P2Y1 c.1622A > G polymorphism affected ADP-induced platelet aggregation in both healthy adults and AMI patients. Simon et al. reported that P2Y1 c.1622A > G was associated with increased platelet aggregation in healthy adults from northern Europe [6], but this result was not confirmed in a Korean population [7]. Patients with CAD carrying the 1622 G/G genotype of the P2Y1 gene had significantly higher levels of arachidonic acid-induced platelet aggregation than noncarriers in Caucasian populations, while another study showed that P2Y1 1622A > G did not contribute to clopidogrel resistance in Indian populations [10,22].
Our results showed that the frequency of the haploid H2 allele was 8.8% in control subjects. This frequency was similar to that in reports on South Indian (8.4%) and Western Indian (8.8%) populations [1,21] but   (Table 6) [7,8,23]. In the present study, the frequency of the haploid H2 allele in the AMI group was significantly higher than that in the control group (P = 0.005). Moreover, the P2Y12 H2 haplotype was positively associated with AMI in the codominant, dominant, and overdominant models (P < 0.05). The association between the P2Y12 H2 haplotype and AMI susceptibility remained significant after adjustment for multiple comparisons by multivariate logistic regression (OR 2.132, 95% CI 1.180-3.854, P = 0.012). Therefore, we conclude that carrying the haploid H2 allele may be one of the risk factors for AMI, which adds very valuable information to this field. Therefore, carrying the H2 haplotype in patients may require closer clinical follow-up. A recent study by Lu et al. [3] showed that carriers with H2 alleles have a higher propensity for cerebral infarction in Sandong, China, and the higher risk of ischemic events in association with the i-744 T > C polymorphism was confirmed in a large meta-analysis summarizing available data up to 2019 [24]. Additionally, several previous studies have demonstrated an association between the P2Y12 H2 haplotype and CA [1,17]. However, a prospective analysis performed on initially healthy American men failed to find a relationship between the P2Y12 H2 haplotype and the risk of MI, IS, and venous thromboembolism [18]. These conflicting results may be due to allelic heterogeneity, case-control selection criteria, and different population backgrounds. In addition, the development of cardiovascular events may be determined by multiple genetic factors and environmental factors [25,26], rather than single-gene polymorphism of P2Y12 [1,27], with regard to the complexity of atherosclerosis etiology. Therefore, it may be crucial to analyze the effects of gene-gene interactions and gene-environment interactions on AMI risk in further studies. Several studies have analyzed the effect of P2Y12 polymorphisms on platelet aggregation with inconsistent results. Prior studies on European and Egyptian populations have shown that the P2Y12 i744T > C polymorphism is associated with enhanced platelet aggregation in response to ADP [5,8]. In a smaller study of 26 AMI patients with a 300 mg dose of clopidogrel from India [21], patients with the P2Y12 H2 haplotype demonstrated a trend toward an impaired response of clopidogrel to inhibit platelet aggregation. Similarly, several studies performed on different populations from Germany [28], Morocco [29], and the Guangzhou Province of China [30] suggested that the P2Y12 H2 haplotype was associated with altered platelet reactivity, thereby resulting in clopidogrel resistance (CR). However, several studies in other ethnicities, such as studies from North India [22], Mexico [19], and Iran [31], have not found any association between the P2Y12 H2 haplotype and CR. The contradictory results in different populations might be partly attributable to racial differences, selection criteria in the study population and methods used to assess platelet aggression. In the present study, we observed that carriers of the P2Y12 H2 haplotype displayed higher ADP-induced MAR than noncarriers among healthy volunteers in Quanzhou, but their association was not confirmed in AMI patients. However, we did not detect the baseline platelet aggregation rate and failed to assess the variability of platelet aggregation in patients before and after AMI development. Additionally, inter individual variations in the platelet aggregation rate may alter the results, and this is one of the limitations of our study. Thus, the effect of the P2Y12 H2 haplotype on platelet aggression needs to be further investigated.
The frequency of the 34 T allele was 20.6%, which is consistent (P > 0.05) with the findings in a Korean population (26.6%) ( Table 6) [7]. Two publications reported that the P2Y12 c.34C > T polymorphism was not associated with platelet aggregation in response to ADP in northern European and Korean populations [5,7]. However, several studies have reported that the P2Y12 c.34C > T polymorphism was associated with a high risk of clopidogrel resistance in Turkish and Han Chinese populations [32,33], but this significant association was not found in a Caucasian population [34]. Our results suggest that P2Y12 c.34C > T polymorphisms do not correlate with the risk of AMI and ADP-induced platelet aggregation.
This study has some limitations. The major limitation is the relatively low number of both AMI patients and control subjects participating in our study. Additionally, a set of gene variations was found to be associated with AMI risk, while our study only assessed the influence of the P2Y1 and P2Y12 genes. Therefore, investigations into more selective genetic variations of the P2Y12 H2 haplotype in large sample sizes must be performed to elucidate the full extent of gene-gene interaction effects on AMI pathogenesis. In addition, further studies should assess the platelet aggregation rate before and after AMI development to calculate the variability of platelet aggregation and investigate its correlation with P2Y1 and P2Y1 polymorphisms.

Conclusion
In this study, we found that the P2Y12 H2 haplotype was associated with a higher risk of AMI. The P2Y12 H2 haplotype was associated with increased ADP-induced platelet aggression in healthy adults, but this association was not confirmed in AMI patients. Thus, the association between the P2Y12 H2 haplotype and platelet aggregation requires further investigation. Additionally, neither the P2Y1 c.1622A > G nor P2Y12 c.34C > T